The present invention relates to humanized monoclonal antibodies and fragments or derivatives thereof which specifically bind carcinoembryonic antigen (CEA), which is an antigen expressed by various human carcinomas including breast, lung, and gastrointestinal carcinomas such as stomach and colon cancers. More specifically, the present invention relates to humanized monoclonal antibodies and humanized antibody fragments and derivatives thereof which are derived from murine monoclonal antibody COL-1, a high affinity anti-CEA antibody. The present invention further relates to methods for producing such humanized monoclonal antibodies specific to CEA, pharmaceutical and diagnostic compositions containing such humanized monoclonal antibodies, and methods of use thereof for the treatment or diagnosis of cancer.
The identification of antigens expressed by tumor cells and the preparation of monoclonal antibodies which specifically bind such antigens is well known in the art. Anti-tumor monoclonal antibodies exhibit potential application as both therapeutic and diagnostic agents. Such monoclonal antibodies have potential application as diagnostic agents because they specifically bind tumor antigens and thereby can detect the presence of tumor cells or tumor antigen in an analyte. For example, use of monoclonal antibodies which bind tumor antigens for in vitro and in vivo imaging of tumor cells or tumors using a labeled form of such a monoclonal antibody is conventional in the art.
Moreover, monoclonal antibodies which bind tumor antigens have well known application as therapeutic agents. The usage of monoclonal antibodies themselves as therapeutic agents, or as conjugates wherein the monoclonal antibody is directly or indirectly attached to an effector moiety, e.g., a drug, cytokine, cytotoxin, etc., is well known.
Essentially, if the monoclonal antibody is attached to an effector moiety, then the monoclonal antibody functions as a targeting moiety, i.e. it directs the effector moiety (which typically possesses therapeutic activity) to the antibody""s target, e.g., a tumor which expresses the antigen bound by the monoclonal antibody. In contrast, when the monoclonal antibody itself operates as a therapeutic agent, the antibody functions both as a targeting moietyxe2x80x94i.e. it will specifically bind a cell which expresses the antigenxe2x80x94and as an effector which mediates therapeutic activity, typically tumor cell lysis. A monoclonal antibody may possess one or more of such effector functions, which include, e.g., antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), among others; these functions are effected by the portion of the antibody molecule generally referred to in the literature as the Fc portion.
One specific tumor antigen to which various monoclonal antibodies have been produced is the carcinoembryonic antigen (CEA). CEA is an antigen complex having a molecular weight of about 180,000 D, which is expressed by numerous carcinomas including gastrointestinal carcinomas, colorectal carcinomas, breast carcinomas, ovarian carcinomas, and lung carcinomas. See, e.g., Robbins et al., Int""l J. Cancer, 53(6):892-897 (1993); Greiner et al., J. Clin. Oncol., 10(5):735-746 (1992); Ohuchi et al., Cancer Res., 47(13):3565-3571 (1987); Muraro et al., Cancer Res., 45(11 Pt. 2):5769-5780 (1985).
The use of monoclonal antibodies to detect various, specific CEA epitopes differentially expressed on human carcinomas has been reported in the literature. See, e.g., Ohuchi et al., Cancer Res., 47(13):3565-3571 (1987); Muraro et al., Cancer Res., 45(11 Pt. 2):5769-5780 (1985).
In particular, Muraro et al. (id.) report generation of monoclonal antibodies designated COL-1 through COL-15, which exhibit a strong, selective reactivity for human colon carcinomas versus normal adult tissues. These antibodies react with distinct, restricted epitopes on CEA. Of these antibodies, the COL-1 antibody has been the focus of considerable attention because of its high affinity for CEA (1.4xc3x97109 Mxe2x88x921) and also because it comprises no detectable reactivity for CEA-related antigens such as the nonspecific cross-treating antigen (NCA) and the normal fecal antigen (NFA). Robbins et al., Int""l J. Canc., 53(6):892-897 (1993).
Because of its binding properties, COL-1 is currently being evaluated for use as a therapeutic agent. For example, Siler et al. (Biotech. Ther., 4(3-4):163-181 (1993)) report the administration of 131I-labeled COL-1 to LS-M4T human colon carcinoma xenograft-containing athymic mice. They report that this treatment resulted in reduction of the rate of tumor growth, within little or no toxicity, and that their results demonstrate the potential therapeutic efficacy of radiolabeled COL-1 in clinical trials. Also, Yu et al. (J. Clin. Oncol., 14(6):1798-1809 (1996)) report that 131I-labeled COL-1 is now in phase 1 clinical trials in patients having gastrointestinal malignancies. They further indicate that the antibody conjugate is well tolerated, except for some hematologic toxicity. In addition, the use of conjugates of COL-1 and xcex2-galactosidase has been shown to specifically kill in vitro tumor cells from a variety of tumor cell lines. Abraham et al.,Cell Biophys., 24-25:127-133 (1994).
However, while murine antibodies, such as COL-1 and other anti-CEA murine antibodies, have applicability as therapeutic agents in humans, they are disadvantageous in some respects. Specifically, because murine antibodies are of foreign species origin, they may be immunogenic in humans. This may result in a neutralizing antibody responsexe2x80x94a human anti-murine antibody (HAMA) responsexe2x80x94which is particularly problematic if the antibodies are desired to be administered repeatedly, e.g., for treatment of a chronic or recurrent disease condition. This is a significant drawback, as some cancer treatments are effected over a prolonged time period, e.g., over several years or longer. Also, because these antibody molecules contain murine constant domains they may not exhibit human effector functions.
In an effort to eliminate or reduce such problems, chimeric antibodies have been disclosed. Chimeric antibodies contain portions of two different antibodies, typically of two different species. Generally, such antibodies contain human constant regions attached to variable regions from another species, typically murine variable regions. For example, some mouse/human chimeric antibodies have been reported which exhibit binding characteristics of the parental mouse antibody and effector functions associated with the human constant region. See, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.; U.S. Pat. No. 4,978,745 to Schoemaker et al.; U.S. Pat. No. 4,975,369 to Beavers et al.; and U.S. Pat. No. 4,816,397 to Boss et al. Generally, these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas. Nishimura et al., Cancer Res., 47:999 (1987). The library is then screened for variable region genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns. Alternatively, cDNA libraries are prepared from RNA extracted from the hybridomas and then screened, or the variable regions are obtained by polymerase chain reaction. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes are then expressed in a cell line of choice, usually a murine myeloma line. Such chimeric antibodies have been used in human therapy.
Moreover, the production of a chimeric mouse anti-human antibody derived from COL-1, which specifically binds CEA, has been reported. See e.g., U.S. Pat. No. 5,472,693 to Gourlie et al. (owned by The Dow Chemical Company).
Also, Morrison et al. report the preparation of several anti-tumor chimeric monoclonal antibodies, in Important Advances in Oncology, Recombinant Chimeric Monoclonal Antibodies, pp. 3-18 (S. A. Rosenberg, ed., 1990) (J. B. Lippincott, Philadelphia, Pa.). Results of clinical trials with chimeric cMAb-17-1A in patients with metastatic colorectal carcinoma now show that this antibody has a 6-fold longer circulation time and significantly reduced immunogenicity as compared to the murine monoclonal antibody from which it was derived. LoBuglio et al., Proc. Natl. Acad. Sci. USA, 86:4220-4224 (1989); Meredith et al., J. Nucl. Med., 32:1162-1168 (1991).
However, while such chimerized monoclonal antibodies typically exhibit lesser immunogenicity, they are still potentially immunogenic in humans because they contain murine variable sequences which may elicit antibody responses. Thus, there is the possibility that these chimeric antibodies may elicit an anti-idiotypic response if administered to patients. Saleh et al., Cancer Immunol. Immunother., 32:185-190 (1990).
Because of the immunogenicity of chimeric antibodies, methods have been developed recently for the production of xe2x80x9chumanizedxe2x80x9d antibodies. Ideally, xe2x80x9chumanizationxe2x80x9d results in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain all the antigen-binding properties of the original antibody, the structure of its combining-site has to be faithfully reproduced in the xe2x80x9chumanizedxe2x80x9d version. This can potentially be achieved by transplanting the combining site of the nonhuman antibody onto a human framework, either: (a) by grafting only the nonhuman complementarity determining regions (CDRs) onto human framework regions (FRs) and constant regions, with or without retention of critical framework residues (see, Jones et al., Nature, 321:522 (1986) and Verhoeyen et al., Science, 239:1534 (1988); or (b) by transplanting the entire nonhuman variable domains (to preserve ligand-binding properties) and also xe2x80x9ccloakingxe2x80x9d them with a human-like surface through judicious replacement of exposed residues (in order to reduce antigenicity) (see, Padlan, Molec. Immunol., 28:489 (1991)).
Essentially, humanization by CDR-grafting involves transplanting only the CDRs onto human framework and constant regions. Theoretically, this should substantially eliminate immunogenicity (except if allotypic or idiotypic differences exist). Jones et al., Nature, 321:522-525 (1986); Verhoeyen et al., Science, 239:1534-1536 (1988), Riechmann et al., Nature, 332:323-327 (1988). However, CDR-grafting by itself may not yield the desired result. Rather, it has been reported that some framework residues of the original antibody may also need to be preserved in order to preserve antigen binding activity. Riechmann et al., Nature, 332:323-327 (1988); Queen et al., Proc. Natl. Acad. Sci. USA, 86:10023-10029 (1989); Tempest et al., Bio. Technology, 9:266-271 (1991); Co et al., Nature, 351:501-502 (1991).
As discussed above, in order to preserve the antigen-binding properties of the original antibody, the structure of its combining site must be faithfully reproduced in the humanized molecule. X-ray crystallographic studies have shown that the antibody combining site is built primarily from CDR residues, although some neighboring framework residues have been found to be involved in antigen binding. Amit et al., Science, 233:747-753 (1986); Colman et al., Nature, 326:358-363 (1987); Sheriff et al., Proc. Natl. Acad. Sci. USA, 84:8075-8079 (1987); Padlan et al., Proc. Natl. Acad. Sci. USA, 86:5938-5942 (1989); Fischmann et al., J. Biol. Chem., 266:12915-12920 (1991); Tulip et al., J. Molec. Biol., 227:122-148 (1992). It has also been found that the structures of the CDR loops are significantly influenced by surrounding framework structures. Chothia et al., J. Molec. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Tramomonteno et al., J. Molec. Biol., 215:175-182 (1990).
In addition to the effect of the framework residues on the CDRs, small but significant differences from the relative disposition of the variable light chain (VL) and variable heavy (VH) domains have been noted (Colman et al., Nature, 326:358-363 (1987)) and those differences are ostensibly due to variations in the residues involved in the interdomain contact (Padlan et al., Molec. Immunol., 31:169-217 (1994)).
Furthermore, structural studies on the effect of the mutation of interior residues, in which changes in side chain volume are involved, have shown that the resulting local deformations are accommodated by shifts in side chain positions that are propagated to distant parts of the molecular interior. This suggests that during humanization the interior residues in the variable domains and in the interface between these domains, or at least the interior volumes, should also be maintained; a humanization protocol in which an interior residue is replaced by one of different properties, such as size, charge, or hydrophobicity, could result in a significant modification of the antigen combining-site structure. One method of potentially identifying the framework residues which need to be preserved is by computer modeling. Alternatively, critical framework residues may potentially be identified by comparing known antibody combining site structures. Padlan, Molec. Immun., 31(3):169-217 (1994).
The residues which potentially affect antigen binding fall into several groups. The first group comprises residues that are contiguous with the combining site surface and which could therefore make direct contact with antigens. These residues include the amino-terminal residues and those adjacent to the CDRs. The second group includes residues that could alter the structure or relative alignment of the CDRs by contacting either the CDRs or the opposite chains. The third group comprises amino acids with buried side chains that could influence the structural integrity of the variable domains. The residues in these groups are usually found in the same positions (id.) according to the adopted numbering system. See Kabat et al., Sequences of Proteins of Immunological Interest, NIH Pub. No. 91-3242 (5th ed., 1991) (U.S. Dept. Health and Human Services, Bethesda, Md.) and Genbank.
Given these effects of changes in amino acid residues, although humanized antibodies are desirable because of their potential low immunogenicity in humans, their production is unpredictable. For example, sequence modification of antibodies may result in substantial or even total loss of antigen binding affinity, or loss of binding specificity. Alternatively, xe2x80x9chumanized antibodiesxe2x80x9d may still exhibit immunogenicity in humans, irrespective of sequence modification.
Thus, there still exists a significant need in the art for novel humanized antibodies to desired antigens. More specifically, there exists a need in the art for humanized antibodies specific to CEA, because of their potential as improved immunotherapeutic and immunodiagnostic agents for treatment and diagnosis of cancers expressing CEA, e.g., gastrointestinal and colorectal cancers, breast cancers, lung cancers, and ovarian cancers, among others.
Toward this end, it is an object of the invention to provide humanized antibodies which are specific to human carcinoembryonic antigen (CEA), i.e. anti-CEA (xe2x80x9cxcex1CEAxe2x80x9d) antibodies. More specifically, it is an object of the invention to provide humanized antibodies derived from murine xcex1CEA antibodies and in particular from COL-1, a specific murine antibody of the IgG2a isotype having high affinity for CEA.
It is also an object of the invention to provide pharmaceutical compositions containing humanized xcex1CEA antibodies. It is a more specific object of the invention to provide pharmaceutical compositions containing humanized antibodies derived from the high affinity murine xcex1CEA antibody, COL-1.
It is another specific object of the invention to provide methods of using humanized xcex1CEA antibodies for treatment of cancers which express CEA, in particular breast, lung, ovarian, gastrointestinal, and colorectal cancers, among others.
It is another object of the invention to provide immunodiagnostic compositions for detecting cancer cells, the compositions containing a humanized xcex1CEA antibody, preferably derived from COL-1, which antibody is in labeled or unlabeled form. It is another object of the invention to provide a method of immunodiagnosis of cancer using compositions which contain a humanized xcex1CEA antibody, preferably derived from COL-1, which is in labeled or unlabeled form.
It is still another object of the invention to provide nucleic acid sequences which encode humanized xcex1CEA antibodies or fragments thereof. It is a more specific object of the invention to provide nucleic acid sequences which encode humanized antibodies derived from the high affinity murine xcex1CEA antibody, COL-1. It is another object of the invention to provide vectors which provide for the expression of humanized xcex1CEA antibodies, in particular humanized antibodies derived from the high affinity murine xcex1CEA antibody, COL-1.